Serological detection of Rickettsia spp. and evaluation of blood parameters in pet dogs and cats from Bangkok and neighboring provinces.

Rickettsiosis is caused by Orientia spp. and Rickettsia spp., arthropod-borne zoonotic intracellular bacteria. The close relationships between pet dogs, cats and owners increase the risk of rickettsial transmission, with limited studies on the seroprevalence in pets. This study investigated the prevalence of rickettsia exposure among dogs and cats in Bangkok and neighboring provinces. The samples from 367 dogs and 187 cats used in this study were leftover serum samples from routine laboratory testing stored at the Veterinary Teaching Hospital. In-house Enzyme-linked immunosorbent assay (ELISA) tests included IgG against the scrub typhus group (STG), typhus group (TG), and spotted fever group (SFG). The seroprevalence in pet dogs was 30.25% (111/367), including 21.53% for STG, 4.36% for TG, and 1.09% for SFG. Co-seroprevalence consisted of 2.72% for STG and TG, 0.27% for STG and SFG, and 0.27% for pangroup infection. The prevalence in cats was 62.56% (117/187), including 28.34% for STG, 4.28% for TG, and 6.42% for STG. Co-seroprevalence in cats consisted of STG and TG (4.28%), STG and SFG (5.35%), TG and SFG (3.21%), and three-group infection (10.69%). No significant difference in seroprevalence for the three serogroups was observed in any of the 64 districts sampled. The mean hematocrit level significantly decreased in seropositive dogs (P<0.05). Seropositive dogs and cats were detected in significantly greater numbers of anemia cases than nonanemia cases (P<0.05) (odds ratio: 7.93, 0.44, p = 0.00, p = 0.01). A significantly higher number of seropositive cats had decreased hemoglobin levels (P<0.05) (odds ratio: 3.63, p = 0.00). The seropositive samples significantly differed among older cats (P<0.05). These high exposures in pet dogs and cats could constitute important relationship dynamics between companion animals and rickettsial vectors. Significantly decreased hematocrit and hemoglobin levels indicated anemia in the exposed dogs and cats. The study findings will raise awareness of this neglected disease among pet owners and veterinary hospital personnel and aid in future public health preventative planning.

Genetic structure and diversity of green turtle (Chelonia mydas) in the Gulf of Thailand.

Background and Aim: The International Union for the Conservation of Nature and Natural Resources lists the green turtle as endangered. Green turtle nesting behavior in the Gulf of Thailand has decreased to <50% of the 1995 level. The population structure of green turtles in the Gulf of Thailand has not yet been studied. This study aimed to characterize the genetic diversity of green turtles in the Gulf of Thailand based on comparisons of mitochondrial DNA (mtDNA) control region with sequences of Indo-Pacific management units (MUs) and rookeries, to investigate population structures, and to explore phylogeographic relationships. Materials and Methods: Blood samples (1 mL each) from 91 stranded green turtles were collected from four parts of the Gulf of Thailand (eastern, upper, central, and lower). The control mtDNA region was amplified by polymerase chain reaction using LCM15382 and H950 primer. The obtained 384-bp or 770-bp sequences were analyzed for haplotype, clade, and haplotype and nucleotide diversities and were used to construct a phylogenetic tree and haplotype network diagram, respectively. In addition, we analyzed genetic differentiation within and among populations of green turtles in the Gulf of Thailand and between green turtles in the Gulf of Thailand and other Indo-Pacific MUs and rookeries. Results: In total, 12 (based on 384 bp) or 13 (based on 770 bp) haplotypes and two clades (clades VII and VIII) were identified, with nine or 10 haplotypes belonging to clade VIII and three haplotypes belonging to clade VII. Of the new haplotypes, four or five were identified and classified as clade VII (two haplotypes, for both fragment lengths) and clade VIII (two or three haplotypes, for 384 bp or 770 bp fragments, respectively). The overall haplotype and nucleotide diversity of green turtles in the Gulf of Thailand were high (0.755 ± 0.039 and 0.01146 ± 0.00248, respectively). Based on the analysis of molecular variance, green turtles in the Gulf of Thailand could be divided into two subpopulations (UC-Eastern Gulf of Thailand [UC-EGT] and lower Gulf of Thailand [LGT]). Comparisons with other MUs and rookeries in the Indo-Pacific showed that UC-EGT was not genetically different from the Peninsular Malaysia and Eastern Taiwan (Lanyu) MUs and the Terrangganu and Mersing rookeries, and LGT were not genetically different from Peninsular Malaysia, Sipadan, Brunei Bay, Eastern Taiwan (Lanyu), Scott Reef and Browse Island, and Gulf of Carpentaria MUs and the Perak, Perhentain Island, Redang, Pahang, and Vietnam rookeries. Conclusion: To the best of our knowledge, this is the first report to identify the haplotypes and clades of green turtles in the Gulf of Thailand and to show that the populations in the Gulf of Thailand not only present high genetic diversity but also have haplotypic endemism. Longer mtDNA fragments (770 bp) increased the resolution of the stock structure. Clade VII is a unique clade not only for Japan but also for Thailand and Malaysia, and CmP82 is a unique haplotype for both the Gulf of Thailand and Malaysia. Conservation and management of these populations are important to preserve the genetic diversity, biological diversity, and evolutionary potential of green turtles in the Gulf of Thailand.

Systematic evaluation of TaqMan real-time polymerase chain reaction assays targeting the dsb and gltA loci of Ehrlichia canis in recombinant plasmids and naturally infected dogs.

Background and Aim: Because of the diversity of local genotypes of Ehrlichia canis, genes targeted by TaqMan real-time polymerase chain reaction (RT-PCR) assays should be systematically evaluated. This study evaluated the amplification efficiency, linearity, precision, and sensitivity of two TaqMan RT-PCR assays targeting the dsb and gltA loci of E. canis in recombinant plasmids and naturally infected dogs. Materials and Methods: Thirty blood samples were collected from dogs showing clinical signs of canine monocytic ehrlichiosis at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The dsb and gltA genes were amplified by conventional PCRs (cPCRs) on the blood samples and were then sequenced. Meanwhile, RT-PCR was used to detect dsb and gltA genes in 10-fold dilutions of the recombinant plasmids. Results: Both dsb and gltA were amplified with a high degree of linearity (R2 ≥0.975 and 0.993, respectively) in all dilutions, although the mean percentage of relative standard deviation of gltA was lower, but the difference was non-significant. The detection limits of RT-PCR and cPCR were 10-7 and 10-6, respectively, for both loci. RT-PCR targeting dsb (22/30; 73.3%) and gltA (15/30; 50%) yielded a number of positive results that did not differ significantly (p=0.06). The RT-PCR positive results of the dsb gene (22/30) differed significantly from that of cPCR (11/30) (p=0.004). In contrast, the RT-PCR positive results of the gltA gene (15/30) did not differ significantly from that of cPCR (12/30) (p=0.43). The mean Ct value (30.2) based on dsb RT-PCR of 22 positive cases was higher than that of gltA RT-PCR (Ct=27.4) on 15 positive cases. The Ct values from dsb RT-PCR were >30 in all seven discordant samples that were not detected by the gltA RT-PCR. Conclusion: RT-PCR targeting the dsb gene was more sensitive for detecting E. canis in naturally infected dogs. This study suggested that TaqMan RT-PCR of the dsb gene should be selected for E. canis research in this region.

Antibacterial Activity of Solanum torvum Leaf Extract and Its Synergistic Effect with Oxacillin against Methicillin-Resistant Staphyloccoci Isolated from Dogs.

Methicillin-resistant staphylococci (MRS) have been considered a veterinary and public health threat that needs to be addressed, as they are known to cause serious infections, with limited therapeutic options. Thus, in this study, we aimed to examine the potential antibacterial activity of the leaf extract of Solanum torvum against MRS isolated from clinically healthy dogs. In total, seven mecA-positive Staphylococcus isolates tested in this study were identified using 16S rRNA gene sequencing, and all of them were classified as multidrug-resistant using disk diffusion tests. According to gas chromatography-mass spectrometry analysis, the main phytochemical components found in the leaf extract were hexadecanoic acid and its ethyl ester and 9,12,15-octadecatrienoic acid, ethyl ester, (Z,Z,Z). The minimum inhibitory concentration (MIC) breakpoints for the leaf extract against all tested isolates ranged from 2 to 16 mg/mL, while the MIC breakpoints for oxacillin were from 2 to 512 mg/L. Although varying effects were found, the positive effects of the leaf extract were most evident in combination with oxacillin. These results suggested that S. torvum leaf extract may complement classical antibiotics and may potentially drive the development of an effective therapeutic option for MRS.

Expression of recombinant 35 kDa fragment of VP2 protein of canine parvovirus using Escherichia coli expression system.

BACKGROUND AND AIM: Canine parvovirus (CPV) is one of the most common viral infections in dogs, causing acute hemorrhagic gastroenteritis and high mortality. Vaccination effectively prevents CPV infection. However, the currently available CPV vaccines have concerns such as maternal immunity interference, shedding of virus vaccine, and false-positive result based on polymerase chain reaction after vaccination. A subunit vaccine can overcome these problems. This study aimed to express the recombinant 35 kDa fragment of the VP2 protein (consisting of epitopes 1-7) and the recombinant full-length VP2 protein (consisting of epitopes 1-10) and to study the ability of these two recombinant proteins to react with rabbit anti-CPV polyclonal antibodies. MATERIALS AND METHODS: The full length and 35 kDa fragment of VP2 gene of CPV were cloned into the pBAD202 Directional TOPO™ expression vector and expressed in E. coli. The recombinant full-length and the recombinant 35 kDa fragment proteins of VP2 were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: The recombinant full-length and the recombinant 35 kDa fragment VP2 genes were successfully cloned and expressed. The optimum concentrations of arabinose and induction time for the recombinant full-length and the recombinant 35 kDa fragment VP2 proteins were 0.2% for 6 h and 0.02% for 6 h, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 protein molecular weights were approximately 81 and 51 kDa, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 proteins specifically interacted with rabbit anti-CPV polyclonal antibodies. CONCLUSION: These results suggest that the recombinant 35 kDa fragment and the recombinant full-length VP2 proteins may be useful in developing a CPV diagnostic test or vaccine.

Molecular identification of Trichuris trichiura and Hymenolepis diminuta in long-tailed macaques (Macaca fascicularis) in Lopburi, Thailand.

BACKGROUND AND AIM: Trichuris trichiura and Hymenolepis diminuta are helminthic intestinal parasites that infect humans and other animals, including non-human primates. However, molecular detection of these parasites remains scarce in long-tailed macaques (Macaca fascicularis), which coexist with human communities in Thailand. Thus, this study aimed to molecularly confirm the occurrence of Trichuris spp. and Hymenolepis spp. infection and determine the species of both parasites that were found in long-tailed macaques. MATERIALS AND METHODS: A total of 200 fecal samples were randomly collected from long-tailed macaques living in Lopburi, Thailand, and tested based on polymerase chain reaction (PCR) assays for Trichuris spp. and Hymenolepis spp. infections. The PCR products were submitted for DNA purification and sequencing. Phylogenetic analysis was performed using the maximum likelihood method. RESULTS: Of 200 tested samples, three (1.5%) were positive for Trichuris spp. Sequence analysis of all positive samples revealed the presence of T. trichiura, while eight samples (8/200, 4%) positive for Hymenolepis spp. were classified as H. diminuta. No significant associations were found between parasite infection and sex of macaques. CONCLUSION: This study revealed that long-tailed macaques harbor T. trichiura and H. diminuta. These results suggested that local residents and tourists must pay attention to limiting contact with long-tailed macaques and take hygienic precautions to reduce the risk of zoonotic and anthroponotic transmission of these parasites between humans and long-tailed macaques.

Detection and genetic characterization of "Candidatus Mycoplasma haemomacaque" infection among long-tailed macaques (Macaca fascicularis) in Thailand using broad-range nested polymerase chain reaction assay.

BACKGROUND AND AIM: Hemoplasmas are defined as small, epicellular parasitic bacteria that can infect the red blood cells of several mammalian species. Diseases caused by these bacteria range from asymptomatic infections to acute hemolytic anemia. However, data on hemoplasmas in non-human primates in Thailand remain to be limited. Therefore, this study aims to determine the occurrence and genetic diversity of hemoplasmas among long-tailed macaques in Thailand. MATERIALS AND METHODS: Blood samples were collected from 339 long-tailed macaques in three provinces of Thailand. DNA was then extracted from the blood samples and tested for hemoplasma using broad-range nested polymerase chain reaction (PCR) based on the 16S rRNA gene. PCR-positive samples were sequenced, and phylogenetic analysis for species identification was conducted. RESULTS: In total, 38 (11.2%) out of the 339 samples were found to be positive for hemoplasmas, based on the broad-range nested PCR assay of the 16S rRNA gene. The 16S rRNA sequences of Mycoplasma spp. were highly similar (98-99% identity) to "Candidatus Mycoplasma haemomacaque." Furthermore, phylogenetic analysis using maximum likelihood demonstrated that the sequences were located in the same cluster of "Ca. M. haemomacaque." CONCLUSION: The detection of hemoplasmas among long-tailed macaques in Thailand is reported. Genetic characterization confirmed that these hemoplasmas are closely related to "Ca. M. haemomacaque." These results indicate that long-tailed macaques in several locations in Thailand may be infected and serve as reservoirs for this parasite.

Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus.

BACKGROUND AND AIM: Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein. MATERIALS AND METHODS: The S1-2 segment of the S gene of the PEDV Thai isolate (G2b) was amplified, cloned into the pBAD202/D-TOPO® vector (Invitrogen, Carlsbad, CA, USA), and expressed in Escherichia coli. The optimum concentration of arabinose and the optimum induction time for the expression of the recombinant S1-2 protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenic reactivity of the recombinant S1-2 protein was determined using Western blot analysis with rabbit polyclonal antibodies against the SM98 strain of PEDV (G1a). RESULTS: The recombinant S1-2 segment of the S gene of the PEDV Thai isolate protein was cloned and the recombinant S1-2 protein was successfully expressed. The optimum concentration of arabinose and the optimum induction time for the induction of the recombinant S1-2 protein were 0.2% and 8 h, respectively. The recombinant S1-2 protein reacted specifically with both rabbit anti-histidine polyclonal antibodies and rabbit anti-PEDV polyclonal antibodies. CONCLUSION: The recombinant S1-2 protein reacted with rabbit anti-PEDV polyclonal antibodies induced by the different PEDV genogroup. Therefore, the recombinant S1-2 protein may be a useful tool for the development of a diagnostic test for PEDV or for a vaccine against PEDV.

Feline panleukopenia virus as the cause of diarrhea in a banded linsang (Prionodon linsang) in Thailand.

A banded linsang (Prionodon linsang) presented at our hospital with clinical signs of acute diarrhea. Fecal samples were positive for canine parvovirus (CPV) as determined by polymerase chain reaction with primers specific for both CPV and feline panleukopenia virus (FPV). The full-length VP2 was cloned, sequenced, and compared with sequences of FPV and CPV strains reported in GenBank. The amino acids that determined the host range were similar to those of FPV. Moreover, amino acid analysis of VP2 revealed over 98% homology to FPV. The FPV isolate was closely related with FPV isolates from Japan, South Korea, and China. To the best of our knowledge, this is the first study to report that banded linsang can be infected with FPV.

Development and application of an indirect enzyme-linked immunosorbent assay for serological survey of Japanese encephalitis virus infection in dogs.

Japanese encephalitis virus (JEV) causes serious acute encephalitis in humans and horses. Although dogs are good sentinels for assessing the risk of JEV infection to humans, a virus neutralization test has been the only method available for measuring the levels of JEV antibody in dogs. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) using purified viral particles as an antigen, was developed for serological survey of JEV infection in dogs. In dogs inoculated experimentally with JEV, the ELISA detected anti-JEV IgM 3 days after infection, with IgM levels peaking 7 days after infection. Anti-JEV IgG was detected 14 days after infection and peaked on 21-28 days after infection. Virus neutralization titers correlated with anti-JEV immunoglobulins measured by the ELISA. To test the utility of the new assay, the seroprevalence of JEV infection among 102 dogs in Kyushu, Japan, was examined by IgG ELISA and by virus neutralization. The correlation coefficient between the IgG ELISA and virus neutralization was 0.813 (p<0.001); comparison of the IgG ELISA and virus neutralization showed a sensitivity and specificity of 82% and 98%, respectively. The IgG ELISA was used to survey dogs in Bangkok, Thailand and 51% of these dogs were found seropositive for JEV. These data suggest that in the capital city of Thailand, the risk of infection with JEV remains high.